The bacterial species Bordetella comprises B. pertussis, B. parapertussis, B. bronchiseptica, and B. avium. The first two microorganisms are human pathogens, while the latter two are generally restricted to non-human hosts. B. pertussis and B. parapertussis cause the disease whooping cough with the former generating more severe symptoms. The disease, or vaccination against the disease (using an inactivated whole-cell vaccine), elicits antibodies against several antigens, typically pertussis toxin (PT), filamentous haemagglutinin (FHA), agglutinogens or fimbriae and the 69 kDa outer membrane protein or pertactin. These proteins represent the major immunogens that may be included, individually or in combination, in any vaccine used to protect against the disease, whether it be the inactivated whole-cell vaccine or a defined component vaccine. Therefore, the efficient expression of these antigens from the vaccine strain is crucial.
During the production of vaccine antigens by fermentation, it has been observed that FHA is secreted at approximately 7 and 10 times the molar level of pertactin and PT, respectively. While protein structural complexity and secretion efficiencies may be important factors influencing antigen yields, the level of expression of B. pertussis antigen genes also may be influenced by the relative strength and the regulation of their respective promoters. Thus, it may be possible to optimize antigen production by substituting autologous promoters, which may either increase or decrease the yield of selected antigens. The resulting B. pertussis strains would be more economical and better immunogens to use directly in whole-cell vaccines. In addition, promoter interchange also may represent a means to enhance fermentation and downstream processing efficiencies for component vaccines by altering the kinetics of production and yields of specific antigens.
Along with other genes, the pertussis toxin operon (TOX), the filamentous haemagglutinin operon (FHA), and the pertactin gene (PRN) are all positively regulated by the Bordetella virulence regulating gene (Bvg), formerly known as VIR. The nucleotide sequences of the TOX, FHA, and PRN structural genes and their promoters have been established and the corresponding protein sequences derived (see below). For the TOX operon, the Bvg responsive region of the promoter has been mapped to a position -170 bp from the start of transcription. The corresponding regulatory regions of the other genes have not yet been determined.
The use of killed whole-cell pertussis vaccines has resulted in a massive reduction in the incidence of whooping cough since their introduction in the 1950s. These vaccine preparations are efficacious but have been known for many years to be reactogenic and to be associated with local and systemic responses in vaccinees. There has thus been a great deal of effort to develop defined acellular pertussis vaccines containing highly purified, well-characterized and non-reactogenic antigens. Such acellular vaccines are used for immunization in Japan and are at various stages of clinical assessment in other countries. Defined pertussis vaccines consist of several combinations of the B. pertussis-specific antigens pertussis toxin (PT), filamentous hemagglutinin (FHA), the 69 kDa outer membrane protein (pertactin) and fimbrial agglutinogens. Replacing whole-cell whooping cough vaccines with the defined acellular preparations has resulted in a substantial increase in the complexity and cost of vaccine manufacture. A major portion of these increased costs is due to the relatively low levels of PT and pertactin produced by B. pertussis strains, even when grown under optimized fermentation conditions. Increasing the level of antigen production by B. pertussis may be achieved by replacing the natural promoter for a gene encoding an antigen by another promoter as described in the present invention.